Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells

Rationale The human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation. Methods and results Here, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2\u27-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the VEGFR-2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network- like structures in the presence of VEGF. Conclusion Chromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tubeforming cells in a 2D matrix in VEGF-dependent manner

Role of Endothelial Nanog in Homeostasis of Adult Tissue Microenvironment

Elevated level of expression of transcription factor (TF) Nanog regulates pluripotency of stem cells. However, the biological significance of low-level expression of Nanog remains unknown. Thus, the goal of this study was to elucidate the role of Nanog in adult vascular endothelial cell (ECs). In view of that, I examined the physiological relevance of quantitative decrease in the expression of Nanog gene in relation to normal EC homeostasis. To examine this, I have used ROSAmT/mG::Nanogfl/+::Cdh5CreERT2 mice to quantitatively reduce the expression of Nanog after tamoxifen (TAM) administration. Here, we incorporated genetic lineage tracing method combined with high-resolution microscopy to visualize green-fluorescent-protein (GFP)-positive ECs in adult vasculature. Loss-of single EC-specific Nanog allele quantitatively reduced VEGFR2 and TERT proteins, and increased EC death. At mechanistic level, ECs stimulated with Wnt3a increased hTERT gene and protein expression; and microscopic analysis showed nuclear accumulation of Nanog and hTERT in response to Wnt3a. Wnt3a also induced Telomerase Activity (TA) in ECs. In a chromatin immunoprecipitation (ChIP) assay, NANOG directly bound to the hTERT promoter; and NANOG transcriptionally activated the hTERT-promoter luciferase reporter activity in ECs. Loss of Nanog in Wnt3a stimulated ECs, reduced hTERT protein level, and reduced luciferase reporter activity. Furthermore, forced expression of NANOG increased expression of hTERT, VEGFR2 and Cyclin-D1 proteins in ECs. Functionally, the loss of single EC-Nanog allele mediated cardiac hypertrophy like phenotype in mouse hearts. Moreover, the re-expression of hTERT into Nanog-depleted ECs partially restored EC survival, proliferation. Together, these series of data uncover a novel mechanism on role of Nanog in the regulation of EC homeostasis in their native tissue microenvironment

NAD(+)-mediated rescue of prenatal forebrain angiogenesis restores postnatal behavior

Intrinsic defects within blood vessels from the earliest developmental time points can directly contribute to psychiatric disease origin. Here, we show that nicotinamide adenine dinucleotide (NAD(+)), administered during a critical window of prenatal development, in a mouse model with dysfunctional endothelial ϒ-aminobutyric acid type A (GABA(A)) receptors (Gabrb3 endothelial cell knockout mice), results in a synergistic repair of impaired angiogenesis and normalization of brain development, thus preventing the acquisition of abnormal behavioral symptoms. The prenatal NAD(+) treatment stimulated extensive cellular and molecular changes in endothelial cells and restored blood vessel formation, GABAergic neuronal development, and forebrain morphology by recruiting an alternate pathway for cellular repair, via previously unknown transcriptional mechanisms and purinergic receptor signaling. Our findings illustrate a novel and powerful role for NAD(+) in sculpting prenatal brain development that has profound implications for rescuing brain blood flow in a permanent and irreversible manner, with long-lasting consequences for mental health outcome

Microscopic analysis of β-catenin, OCT4, N-cadherin and vWF in epigenetically modified cells.

<p>hADFCs were plated on coverslips, left untreated or treated with epigenetic modifiers as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176496#pone.0176496.g001" target="_blank">Fig 1A</a>, and stained with anti-β-catenin (A-D, green) or anti-OCT4 (E-P, green) or anti-vWF (Q-T, red). Representative microscopic images of: <b>A&E</b>) d2 control untreated cells; <b>B&F</b>) d3 cells treated once with Aza + TSA; <b>C&G</b>) d4 cells received two doses of Aza + TSA; <b>D&H</b>) d5, treated with a third dose of Aza + TSA and TDG. White arrowheads indicate β-catenin; magenta arrowheads show nuclear accumulation of OCT4. <b>I-L</b>) d5 cells treated with a third dose of Aza + TSA and TDG were stained with DAPI (blue), TRITC-phalloidin (red), and OCT4 (green). Magenta arrows (<b>L</b>) indicate nuclear localization. <b>M-P</b>) d5 cells treated with a third dose of Aza + TSA and TDG were stained with DAPI (blue), OCT4 (green), and N-cadherin (red). <b>Q-T</b>) d2, d3, d4 and d5 hADFC were stained with DAPI (blue) and vWF (red). Magnification is as shown.</p

A model of the conversion process of fibroblast cells into tube-forming cells.

<p>hADFCs are mature differentiated cells with highly condensed chromatin containing histones tightly bound to DNAs. However, upon treatment with Aza and TSA for two or three cycles, histone acetylation increases, DNA methylation decreases, the chromatin becomes relaxed, so that the transcription factors could activate the expression of mesodermal-endothelial genes. These events mediate the formation of tube-like structures in 2D Matrigel in a VEGF-dependent manner.</p

Chromatin modification strategy, timeline of experiments, and gene expression analyses.

<p><b>A</b>) The strategy and timeline used for all the experiments described in this study. <b>B</b>) Descriptions of the four experimental groups: (<b>a</b>) day-2 (d2) dish is control untreated cells; (<b>b</b>) day-3 (d3) dish received one dose of Aza + TSA for 24 hours before the cells were used; (<b>c</b>) day-4 (d4) received a second dose of Aza + TSA for 24 hours before use; (d) day-5 (d5) received a third dose of Aza + TSA and TDG 24 hours before use. <b>C</b>) Total mRNAs prepared from these cells were subjected to q-RT-PCR using gene-specific primers for <i>18S RNA</i> (control), <i>VEGFR-2/FLK1</i>, <i>OCT4</i>, and <i>LPP3</i>. Experiments were carried out three times, with triplicate mRNAs. * p<0.05 or ** p<0.01 vs. control <i>18S RNA</i> from same group.</p

Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells

<div><p>Rationale</p><p>The human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation.</p><p>Methods and results</p><p>Here, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2'-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the <i>VEGFR-2/FLK1</i> promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network-like structures in the presence of VEGF.</p><p>Conclusion</p><p>Chromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tube-forming cells in a 2D matrix in VEGF-dependent manner.</p></div

Chromatin modification mediate expression of mesodermal and endothelial cells marker proteins.

<p>Total protein extracts prepared from: a) d2 control cells; b) d3 cells treated once with Aza + TSA; c) d4 cells treated twice with Aza + TSA; d) d5, treated with a third dose of Aza + TSA and TDG were resolved by denaturing SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and analyzed by Western blotting (WB) with antibodies to: <b>A</b>) β-catenin, <b>B</b>) OCT4, and <b>C</b>) VEGFR-2/FLK1; <b>D</b>) Brachyury; <b>E</b>) N-cadherin; <b>F</b>) TIE-2; <b>G</b>) CD31; <b>H</b>) LPP3; and (<b>I</b>) β-tubulin. β-tubulin antibody was used to judge equal loading of proteins across the lanes. (<b>J</b>) Representative image of ponceau-S stained nitrocellulose membrane. Molecular weights are given in kiloDaltons (kDa). The numbers at the lowermost rows of each image indicate signal intensities that were quantified by NIH ImageJ. Experiments were carried out three times.</p

In epigenetically modified hADFCs OCT4 binds to the <i>VEGFR-2/FLK1</i>-promoter sequence.

<p><b>A)</b> Schematic of the human <i>VEGFR-2/FLK1</i>-promoter region. The approximate location of the forward (For) and reverse (Rev) primers used to amplify the ~860-bp PCR product upstream of the transcription start site (TSS) is shown. <b>B</b>) Primers used for amplifying OCT4 binding region in <i>VEGFR2/FLK1</i> promoter. <b>C</b>) Representative ethidium bromide (EtBr) stained agarose gel showing PCR product obtained from anti-OCT4 ChIP experiment using chromatin from <b>a</b>) d2 control untreated cells; <b>b</b>) d3 cells treated once with Aza + TSA; <b>c</b>) d4 cells treated twice with Aza + TSA; <b>d</b>) d5, treated with a third dose of Aza + TSA and TDG. <b>D</b>) Strategy, timeline of Aza + TSA treatment and OCT4-knockdown. <b>E</b>) Total cell extracts prepared from: d2 control hADFCs; d2 cells receiving Aza and TSA; d3 cells receiving second dose of Aza and TSA; d2/kd, d2 cells receiving Aza and TSA were subjected to <i>OCT4</i>-shRNA-knockdown (d2/kd); d3 cells receiving second dose of Aza and TSA were subjected to <i>OCT4</i>-shRNA-knockdown (d3/kd) were subjected to WB with indicated antibodies. GAPDH was used to judge equal loading of proteins across the lanes. The numbers below the OCT4 and VEGFR-2 WB panels represent WB signal intensities. Experiments were repeated three times.</p

Human gene specific primers used for q-RT-PCR data shown in S1 Fig.

<p>Human gene specific primers used for q-RT-PCR data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176496#pone.0176496.s001" target="_blank">S1 Fig</a>.</p